The goal of this project is to define the molecular mechanisms involved in the replication of mammalian retroviruses and in particular, to understand the factors which influence the regulated expression of viral genetic information. Studies are being carried out on the functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT). We have been investigating the effect of replacing the viral RNase H domain with a cellular RNase H on processive DNA synthesis, using templates having sequences from the polypurine tract, an essential region at the 3'-end of the viral genome. Two chimeric RTs with the entire murine leukemia virus (MuLV) polymerase domain or all but the last 19 amino acids, respectively, fused to E. coli RNase H have been expressed and purified. No major differences in the activities of the two chimeric RTs are detected, indicating that the additional 19 amino acids, which form the C-terminus of the connection subdomain, do not significantly influence the enzyme function of these mutants. The chimeric RTs as well as an RT mutant missing the entire RNase H domain are deficient in catalyzing processive DNA synthesis, although the effect is more pronounced with the RNase H-minus mutant. The results of band shift assays indicate that this defect reflects inefficient binding of the mutant RTs to primer-template. Moreover, unlike wild-type RT, which appears to be active in a dimeric form during DNA synthesis, mutant reactions contain mostly RT monomers. We conclude from these findings that (i) the presence of a wild-type polymerase domain is not sufficient by itself to ensure processive DNA synthesis; (ii) the viral RNase H domain stabilizes RT binding to primer-template, which in turn facilitates formation of the active form of RT and processive DNA synthesis; and (iii) an RT with a heterologous RNase H is more processive than an RT without any RNase H domain. Other studies on MuLV RT involve genetic and biochemical approaches to explore the influence of the connection subdomain on polymerase and RNase H activities and to investigate factors which affect enzyme pausing during primer extension. Studies on HIV-1 RT are focusing on the requirements for site-specific cleavage at the 3'-end of the polypurine tract and initiation of plus-strand viral DNA synthesis. Sequence requirements for in vitro dimerization of viral genomic RNA and the role of nucleocapsid protein in this process are also being investigated.